Dmitri Demidov

Identification and Dynamics of Two Classes of Aurora-Like Kinases in Arabidopsis and Other Plants

Aurora-like kinases play key roles in chromosome segregation and cytokinesis in yeast, plant, and animal systems. Here, we characterize three Arabidopsis thaliana protein kinases, designated AtAurora1, AtAurora2, and AtAurora3, which share high amino acid identities with the Ser/Thr kinase domain of yeast Ipl1 and animal Auroras. Structure and expression of AtAurora1 and AtAurora2 suggest that these genes arose by a recent gene duplication, whereas the diversification of plant α and β Aurora kinases predates the origin of land plants. The transcripts and proteins of all three kinases are most abundant in tissues containing dividing cells. Intracellular localization of green fluorescent protein–tagged AtAuroras revealed an AtAurora-type specific association mainly with dynamic mitotic structures, such as microtubule spindles and centromeres, and with the emerging cell plate of dividing tobacco (Nicotiana tabacum) BY-2 cells. Immunolabeling using AtAurora antibodies yielded specific signals at the centromeres that are coincident with histone H3 that is phosphorylated at Ser position10 during mitosis. An in vitro kinase assay demonstrated that AtAurora1 preferentially phosphorylates histone H3 at Ser 10 but not at Ser 28 or Thr 3, 11, and 32. The phylogenetic analysis of available Aurora sequences from different eukaryotic origins suggests that, although a plant Aurora gene has been duplicated early in the evolution of plants, the paralogs nevertheless maintained a role in cell cycle–related signal transduction pathways.

The temporal and spatial pattern of histone H3 phosphorylation at serine 28 and serine 10 is similar in plants but differs between mono- and polycentric chromosomes

Immunolabeling using site-specific antibodies against phosphorylated histone H3 at serine 10 or serine 28 revealed in plants an almost similar temporal and spatial pattern of both post-translational modification sites at mitosis and meiosis. During the first meiotic division the entire chromosomes are highly H3 phosphorylated. In the second meiotic division, like in mitosis, the chromosomes contain high phosphorylation levels in the pericentromeric region and very little H3 phosphorylation along the arms of monocentric species. In the polycentric plant Luzula luzuloides phosphorylation at both serine positions occurs along the whole chromosomes, whereas in monocentric species, only the pericentromeric regions showed strong signals from mitotic prophase to telophase. No phosphorylated serine 10 or serine 28 was detectable on single chromatids at anaphase II resulting from equational segregation of rye B chromosome univalents during the preceding anaphase I. In addition, we found a high level of serine 28 as well as of serine 10 phosphorylation along the entire mitotic monocentric chromosomes after treatment of mitotic cells using the phosphatase inhibitor cantharidin. These observations suggest that histone H3 phosphorylation at serine 10 and 28 is an evolutionarily conserved event and both sites are likely to be involved in the same process, such as sister chromatid cohesion.

Arabidopsis α Aurora Kinases Function in Formative Cell Division Plane Orientation

This work reports on a genetic approach to unravel the developmental function of the group α Aurora kinases by analyzing a viable double mutant. The results show that α Aurora kinases are functionally divergent from the β Aurora kinase and suggest that α Aurora kinases regulate formative division plane orientation throughout development. To establish three-dimensional structures/organs, plant cells continuously have to adapt the orientation of their division plane in a highly regulated manner. However, mechanisms underlying switches in division plane orientation remain elusive. Here, we characterize a viable double knockdown mutant in Arabidopsis thaliana group α Aurora (AUR) kinases, AUR1 and AUR2, (aur1-2 aur2-2), with a primary defect in lateral root formation and outgrowth. Mutant analysis revealed that aur1-2 aur2-2 lateral root primordia are built from randomly oriented cell divisions instead of distinct cell layers. This phenotype could be traced back to cytokinesis defects and misoriented cell plates during the initial anticlinal pericycle cell divisions that give rise to lateral root primordia. Complementation assays showed that the Arabidopsis α group Aurora kinases are functionally divergent from the single β group member AUR3 and that AUR1 functions in division plane orientation prior to cytokinesis. In addition to defective lateral root patterning, aur1-2 aur2-2 plants also show defects in orienting formative divisions during embryogenesis, divisions surrounding the main root stem cell niche, and divisions surrounding stomata formation. Taken together, our results put forward a central role for α Aurora kinases in regulating formative division plane orientation throughout development.

Arabidopsis KINETOCHORE NULL2 Is an Upstream Component for Centromeric Histone H3 Variant cenH3 Deposition at Centromeres

This work finds that Arabidopsis KINETOCHORE NULL2 (KNL2) colocalizes with the centromere histone variant cenH3. Characterization of knl2 mutants showed reduction of cenH3 deposition at centromeres, abnormalities of mitosis and meiosis, seed abortion, and alterations in DNA methylation. The centromeric histone H3 variant cenH3 is an essential centromeric protein required for assembly, maintenance, and proper function of kinetochores during mitosis and meiosis. We identified a KINETOCHORE NULL2 (KNL2) homolog in Arabidopsis thaliana and uncovered features of its role in cenH3 loading at centromeres. We show that Arabidopsis KNL2 colocalizes with cenH3 and is associated with centromeres during all stages of the mitotic cell cycle, except from metaphase to mid-anaphase. KNL2 is regulated by the proteasome degradation pathway. The KNL2 promoter is mainly active in meristematic tissues, similar to the cenH3 promoter. A knockout mutant for KNL2 shows a reduced level of cenH3 expression and reduced amount of cenH3 protein at chromocenters of meristematic nuclei, anaphase bridges during mitosis, micronuclei in pollen tetrads, and 30% seed abortion. Moreover, knl2 mutant plants display reduced expression of suppressor of variegation 3-9 homologs2, 4, and 9 and reduced DNA methylation, suggesting an impact of KNL2 on the epigenetic environment for centromere maintenance.

Novel phosphorylation of histone H3 at threonine 11 that temporally correlates with condensation of mitotic and meiotic chromosomes in plant cells.

A novel mitosis-specific phosphorylation site in histone H3 at threonine 11 has been described for mammalian cells. This modification is restricted to the centromeric region while phosphorylation at the classical H3 sites, Ser10 and Ser28 occurs along the entire chromosomal arms. Using phosphorylation state-specific antibodies we found that phosphorylation at threonine 11 occurs also in plant cells, during mitosis as well as meiosis. However, in contrast to animal cells, ph(Thr11)H3 was distributed along the entire length of condensed chromosomes, whereas H3 phosphorylated at Ser10 and Ser28 appeared to be restricted to centromeric/pericentromeric chromatin. Phosphorylation at Thr11 started in prophase and ended in telophase, it correlated with the condensation of mitotic and meiotic chromosomes and was independent of the distribution of late replicating heterochromatin and Giemsa-banding positive regions. Interestingly, treatment of cells with the phosphatase inhibitor cantharidin revealed a high level of Thr11 phosphorylation in interphase cells, in this case particularly in pericentromeric regions. These data show that histone modifications are highly dynamic. Moreover, animal and plant organisms may have evolved individual histone codes.

Distribution patterns of phosphorylated Thr 3 and Thr 32 of histone H3 in plant mitosis and meiosis

Cell cycle dependent phosphorylation of conserved N-terminal tail residues of histone H3 has been described in both animal and plant cells. Through cytogenetic approaches using different plant species we show a detailed description of distribution patterns of phosphorylated histone H3 at either threonine 3 or threonine 32 in mitosis and meiosis. In meristematic cells of the large genome species Secale cereale, Vicia faba and Hordeum vulgare we have found that phosphorylation of both threonine residues begins in prophase, and dephosphorylation occurs in late anaphase. However, in the small genome species Arabidopsis thaliana dephosphorylation occurs at anaphase. In the first division of meiosis of species with large genomes phosphorylation of histone H3 at either threonine 3 or threonine 32 is seen first in diakinesis and extends to anaphase I, whereas in the second division these post-translational modifications are visible at metaphase II through anaphase II. While in A. thaliana dephosphorylation takes place at anaphase I and II. In all species analysed phosphorylated H3 at either threonine 3 or threonine 32 are distributed along the entire length of chromosomes during mitotic metaphase and metaphase I. In the second meiotic division threonine 3 phosphorylation is restricted to the pericentromeric domain, while phosphorylation of threonine 32 is widespread along chromosome arms of all species analysed.

Point mutation impairs centromeric CENH3 loading and induces haploid plants

Significance The generation of haploids is the most powerful means to accelerate the plant-breeding process. We elucidated whether point mutations in the centromere-specific histone H3 variant CENH3 could be harnessed for the induction of haploids. We identified plants with impaired centromere loading caused by a mutation in the centromere-targeting domain (CATD). The same mutation results in reduced loading of CENH3 in transgenic Arabidopsis and sugar beet. Arabidopsis plants carrying this single point mutation in wild-type CENH3 were used as haploid inducers. Because the identified mutation site is highly conserved and because point mutations can be generated by mutagenesis or genome editing, the described method offers opportunities for application in a wide range of crop species. The chromosomal position of the centromere-specific histone H3 variant CENH3 (also called “CENP-A”) is the assembly site for the kinetochore complex of active centromeres. Any error in transcription, translation, modification, or incorporation can affect the ability to assemble intact CENH3 chromatin and can cause centromere inactivation [Allshire RC, Karpen GH (2008) Nat Rev Genet 9 (12):923–937]. Here we show that a single-point amino acid exchange in the centromere-targeting domain of CENH3 leads to reduced centromere loading of CENH3 in barley, sugar beet, and Arabidopsis thaliana. Haploids were obtained after cenh3 L130F-complemented cenh3-null mutant plants were crossed with wild-type A. thaliana. In contrast, in a noncompeting situation (i.e., centromeres possessing only mutated or only wild-type CENH3), no uniparental chromosome elimination occurs during early embryogenesis. The high degree of evolutionary conservation of the identified mutation site offers promising opportunities for application in a wide range of crop species in which haploid technology is of interest.

AtHaspin phosphorylates histone H3 at threonine 3 during mitosis and contributes to embryonic patterning in Arabidopsis.

Post-translational histone modifications regulate many aspects of chromosome activity. Threonine 3 of histone H3 is highly conserved, but the significance of its phosphorylation is unclear, and the identity of the corresponding kinase in plants is unknown. Therefore, we characterized the candidate kinase in Arabidopsis thaliana, called AtHaspin. Recombinant AtHaspin in vitro phosphorylates histone H3 at threonine 3. Reduction of H3 threonine 3 phosphorylation level and reduced chromatin condensation in interphase nuclei by AtHaspin RNAi supports the proposition that this kinase is involved in histone H3 phosphorylation in vivo in mitotic cells. In addition, we provide a developmental function for a Haspin kinase. At the whole plant level, altered expression of the kinase induced pleiotropic phenotypes with defects in floral organs and vascular tissue. It reduced fertility and modified adventitious shoot apical meristems that then gave rise to plants with multi-rosettes and multi-shoots. Haspin mutant embryos frequently showed alteration in division plane orientation that could be traced back to the earliest divisions of embryo development, thus Haspin contributes to embryonic patterning.

Anti-Phosphorylated Histone H2AThr120: A Universal Microscopic Marker for Centromeric Chromatin of Mono- and Holocentric Plant Species

Based on the analysis of 20 different monocot and eudicot species, we propose that the centromeric distribution of the phosphorylated histone H2AThr120 is evolutionary highly conserved across species with mono- and holocentric chromosomes. Therefore, antibodies recognizing the phosphorylated threonine 120 of the histone H2A can serve as a universal marker for the cytological detection of centromeres of mono- and holokinetic plant species. In addition, super resolution microscopy of signals specific to the centromere-specific histone H3 variant CENH3 and to H2AThr120ph revealed that these histone variants are incorporated into different nucleosomes, which form distinct, partly intermingled chromatin domains. This specific arrangement of both histone variants suggests different centromeric functions during the cell cycle.

Cytomixis doesn’t induce obvious changes in chromatin modifications and programmed cell death in tobacco male meiocytes

Cytomixis is a poorly studied process of nuclear migration between plant cells. It is so far unknown what drives cytomixis and what is the functional state of the chromatin migrating between cells. Using immunostaining, we have analyzed the distribution of posttranslational histone modifications (methylation, acetylation, and phosphorylation) that reflect the functional state of chromatin in the tobacco microsporocytes involved in cytomixis. We demonstrate that the chromatin in the cytomictic cells does not differ from the chromatin in intact microsporocytes according to all 14 analyzed histone modification types. We have also for the first time demonstrated that the migrating chromatin contains normal structures of the synaptonemal complex (SC) and lacks any signs of apoptosis. As has been shown, the chromatin migrating between cells in cytomixis is neither selectively heterochromatized nor degraded both before its migration to another cell and after it enters a recipient cell as micronuclei. We also showed that cytomictic chromatin contains marks typical for transcriptionally active chromatin as well as heterochromatin. Moreover, marks typical for chromosome condensation, SC formation and key proteins required for the formation of bivalents were also detected at migrated chromatin.